Additional file 1 of Multiple paths to cold tolerance: the role of environmental cues, morphological traits and the circadian clock gene vrille

Additional file 1: Table S1. Information on fly collecting sites and years, and the exact coordinates (latitude, longitude) and altitudes for each collecting site. Table S2. A List of 19 bioclimatic variables used in the PCA (WorldClim database v2.1, 2.5 min spatial resolutions; current data 1970–2000; Fick and Hijmans 2017; www.worldclim.org ). Table S3. 19 bioclimatic variables for each site were extracted from WorldClim database v2.1. Table S4. Principal components with their variance, cumulative variance and Eigenvalues. Table S5. Contributions (loadings) of the altitude and 19 bioclimatic variables on the Principal Component (PC). Table S6. The best-fit model for CCRT, CTmin, body colour and size of D. montana and D. flavomontana was defined based on Akaike Information Criterion (AIC). Table S7. Summary of the best-fit model results on the effects of latitude and/or climatic factors (PC1) on cold tolerance and body colour of D. flavomontana. Table S8. Summary of the correlations between two distance matrices. Table S9. Summary of the effect of treatment (LacZ and no-injection controls and RNAi with vrille) on the expression levels of vrille. Table S10. Summary of the effects of cold acclimation treatment on cold tolerance. Table S11. Summary of the effects of silencing vrille gene on cold tolerance. Table S12. The number of pairwise nucleotide differences in vrille exons among D. montana and D. flavomontana populations. Table S13. The number of pairwise amino acid differences in translated vrille exons among D. montana and D. flavomontana populations. Table S14. Pairwise differences in the mean CTmin among D. montana and D. flavomontana populations. Table S15. Pairwise differences in the mean CCRT among D. montana and D. flavomontana populations. Table S16. Quantitative real time PCR (qPCR) primers and their efficiencies (%) for vrille gene and reference genes (Tub2, Rpl32), and primers designed for dsRNA used in RNA interference (RNAi). Figure S1. The effectiveness of RNAi was investigated 12, 24 and 48 h after injections. Figure S2. Preliminary colour intensity measurements of different D. montana and D. flavomontana populations. Figure S3. The effect of the freezing time (in months) on body weight. Figure S4. Distributions and Shapiro–Wilk test statistics and P-values for testing normality of CTmin, CCRT, body colour and body size (measured as weight) data of D. montana and D. flavomontana. Figure S5. vrille sequence alignment and annotation of eight D. montana samples originating across study sites. Figure S6. vrille sequence alignment and annotation of eight D. flavomontana samples originating across study sites. Figure S7. Distributions and Shapiro–Wilk test statistics and P-values for testing normality of CTmin and CCRT data of D. montana in RNAi studies.